Cells communicate with each other by a variety of signal molecules, such as neurotransmitters, hormones and growth factors that are detected by specific receptors on the plasma membrane of the responding cell. Stimulation of a receptor by its agonist initiates a series of biochemical processes that produce an intracellular signal called a second messenger. This second messenger ultimately causes a change in the behavior of an cell resulting in a biological response such as secretion of an enzyme, contraction or initiation of cell division.
The cyclic nucleotides cyclic adenine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP) act as second messengers in many cell types to couple extracellular stimulatory or inhibitory signals with the appropriate cellular physiological responses. Examples of important cellular physiological responses that are regulated by cyclic nucleotide second messengers include visual excitation, activation of adenylate cyclase and growth hormone secretion by growth hormone releasing factor, regulation of ion channels by neurotransmitters and membrane receptor activation and olfaction. See, for example, Gilman, Ann. Rev. Biochem., 56:615-49 (1987); Neer et al., Nature, 333:129-134 (1988); Vallar et al., Nature, 330:566-568 (1987); Dolphin, Trends in Neurosciences, Volume 10, February (1987); and Stryer, Ann. Rev. Neurosci., 9:87-119 (1986).
The hormone-responsive tissues are the most extensively studied examples of regulation of physiological responses by cyclic nucleotides. For example, growth hormone releasing factor (GRF) is produced by the hypothalamus, and binds to a receptor on growth hormone (GH)-producing pituitary somatotrope cells. This binding causes stimulation of the ubiquitous receptor-associated G-protein G.sub.s, leading to activation of adenylate cyclase which in turn causes the elevation of cAMP levels within the somatotrope cells. Bilezikjian et al., Endocrinology. 113 1726-1731 (1983) and Vallar et al., Nature, 330:566-568 (1987). The elevated cAMP levels within the somatotrope cells increases the rate of growth hormone gene transcription and consequently the secretion of growth hormone from the somatotrope cells. Schofield, Nature, 215:1382-1383 (1967) and Barinaga et al., Nature, 314:279-281 (1985). The somatotrope cells also proliferate in response to the elevated cAMP level. Billesterup et al., Proc. Natl. Acad. Sci., U.S.A. 83:6854-6857 (1986). Other G-protein dependent cascades with different specific stimulatory or inhibitory outcomes are found in other hormone-responsive tissues such as the adrenal cortex, thyroid and gonads, as well as in various subsets of neurotransmitter- or light-responsive neurons such as the retinal photoreceptors.
Several bacterial toxins are known to perturb the level of second messengers in cells. See for example, Gill et al., J. Infect. Dis., 133:S103-S107 (1976); Gill et al., Proc. Natl. Acad. Sci., U.S.A., 1975:3050-054 (1978); Katada et al., J. Biol. Chem., 257:7210 (1982). These bacterial toxins activate or inactivate the G-proteins by catalyzing the transfer of an adenosine diphosphate (ADP) ribose group from nicotinamide adenine dinucleotide (NAD) to an amino acid located in or near with the guanine triphosphate (GTP) binding site of the G-protein. Dolphin, Trends in Neurosciences, 10:53-57 (1987). Since the toxins act enzymatically, the entry of a single toxin molecule into the cell can, in principle, ADP-ribosylate all of its substrates within the cell. Yamiazumi et al., Cell, 15:245-250 (1978). Examples of G-protein ribosylating toxins include the exotoxins from Escherichia coli. Vibrio cholera, and Bordetella pertussis. Pappenheimer, J. Hyg., 93:397-404 (1984).
All of these toxins together with several other plant and bacterial toxins appear to have a similar structure, consisting of at least one A chain disulfide-linked to at least one B chain. The B-chains bind to specific glycoprotein receptors on the cell surface and allow entry of the A-chain which contains the enzymatic activity into the cell. Ui et al., in Pertussis Toxin, Sekura et al., eds., 19-43, Academic Press, San Diego (1985) and Gill, Proc. Natl. Acd. Sci. U.S.A., 72:2064-2068 (1975).
Recent advances in transformation technology allow the introduction of a specific gene sequence into the germ-line of mammals. For review, see Palmiter and Brinster, Ann. Rev. Genet., 20:465-499 (1986). The genes introduced using this technology are integrated into the germ-line and therefore pass from one generation of the next. These techniques have been used to express a variety of end product proteins, such as hormones and to specifically kill certain cells within the developing mammal. Evans et al., U.S. Pat. No. 4,870,009, Sep. 26 (1989); and Palmiter et al., Cell, 50:435-443 (1987).
However, the transgenic mammals produced to date have expressed only end product proteins such as hormones, immunoglobulins, and other structural proteins. Expression of these and similar products only allows the elevation of one protein product or the elevation of a series of products within a given biochemical pathway of the cells within the transgenic mouse. Other studies that have specifically killed or ablated a given cell lineage within the transgenic animals have allowed the study of the effects of the cell lineage's absence but do not allow the manipulation of the multiple protein products and the physiology produced from the stimulation of a given cell lineage within the transgenic mouse.